Overview

  • Product name
    Anti-Pax2 antibody [EP3251]
    See all Pax2 primary antibodies
  • Description
    Rabbit monoclonal [EP3251] to Pax2
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, Flow Cyt, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Pax2 aa 1-100. The exact sequence is proprietary.
    Database link: Q02962
    (Peptide available as ab188213)

  • Positive control
    • WB: Human fetal kidney tissue lysate IHC-P: Human fetal kidney, human normal kidney, human renal cell carcinoma, mouse liver and rat cerebral cortex tissues. ICC/IF: HEK293 cells. Flow Cyt: HEK293 and K562 cells.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab79389 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000 - 1/10000. Detects a band of approximately 45 kDa (predicted molecular weight: 45 kDa).Can be blocked with Pax2 peptide (ab188213).
Flow Cyt 1/50.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IHC-P 1/500 - 1/2500.

See IHC antigen retrieval protocols.

ICC/IF 1/50.

Target

  • Relevance
    Pax2 is a transcription factor critically required during the development of the nervous and excretory systems, including the midbrain, hindbrain, spinal cord, eye, ear and urogenital tract. Like other products of the Pax gene family, Pax2 encodes a conserved 128 amino acid paired box DNA-binding domain in the N-terminal portion of the molecule. Function: Probable transcription factor that may have a role in kidney cell differentiation. Has a critical role in the development of the urogenital tract, the eyes, and the CNS. Tissue specificity: Expressed in primitive cells of the kidney, ureter, eye, ear and central nervous system.
  • Cellular localization
    Nuclear
  • Database links
  • Alternative names
    • FSGS7 antibody
    • Paired box 2 antibody
    • Paired box gene 2 antibody
    • paired box homeotic gene 2 antibody
    • paired box protein 2 antibody
    • Paired box protein Pax 2 antibody
    • Paired box protein Pax-2 antibody
    • Paired box protein Pax2 antibody
    • PAPRS antibody
    • Pax 2 antibody
    see all

Images

  • Pax2 is expressed in melanocytes of benign nevi (A) and melanoma cells of patients with malignant melanoma (B).

    Cells were grown on coverslips and fixed with 4% paraformaldehyde/PBS. After washing the cells with PBS, cells were permeabilized and blocked with 0.1% Triton X-100/PBS containing 5% BSA. Pax2 (green) was examined by immunofluorescent analysis using ab79389 at 1/100 dilution (incubated for 1 hour at room temperature). Following 3 times washing, bound antibodies were deteced by Alexa 488 conjugated goat anti-mouse or Cy3 conjugated goat anti-mouse secondary antibodies.

    Following PBS-washing nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI, Blue) and cells were mounted in Fluoromount-G™ and examined by fluorescence microscopy.

    White arrows in the higher magnified insets indicate PAX2 expression in nucleoli of melanocytes of benign nevi (A), yellow arrows in the higher magnified insets specify PAX2 expression in nucleoli of melanoma cells (B).

  • Immunohistochemical analysis of Pax2 expression in tissue sections of benign nevi and malignant melanoma using ab79389 at 1/100 dilution.

    All specimens were fixed in 4% formaline (pH 7.4), embedded in paraffin followed by cutting with a microtome (3 µm thickness). The slides were deparaffinized in xylol for 20 minutes and then rehydrated in descending series of ethanol (100%, 100%, 96%, 96%, 70%, and 70%). For antigen retrieval the slides were boiled in citrate buffer (pH 6.0) for 40 min, and then allowed to cool down for 15 min. After washing with PBS buffer the endogenous peroxidase was blocked with H2O2 for 15 min at room temperature. After washing in PBS the slides were incubated with the antibody against PAX2 (dilution 1∶100) for 60 min at room temperature and washed in PBS again.

    The secondary antibody was incubated for 20 min at room temperature and after washing the slides in PBS the biotin streptavidine label was incubated for 20 min at room temperature. A detection kit including horseradish peroxidase and diaminobenzidine as chromogene was applied for 5 min. Counterstaining was performed with hematoxilin for 6 min.

    (A) In normal sweat glands, Pax2 is expressed in gland epithelial cells (black arrows) while intermingled stromal cells only show very weak or absent nuclear Pax2 expression (green arrow) Bar represents 100 µm.

    (B, C) Normal appearing epidermal cell layers adjacent to (B, bar represent 100 µm) nevi or (C, bar represent 200 µm) malignant melanoma show a differentially Pax2 expression with strongest Pax2 levels in germinal basal cell layers (black arrows) decreasing in higher differentiated keratinocytes and finally being absent in corneocytes (green arrows).

    (D) Malignant melanoma cells - heterogeneous nuclear Pax2 expression. Strongest expression in large atypical nuclei with prominent nucleoli (black arrows). 

    (E, F) Pax2 expression in intradermal nevi was heterogeneous and did not correlate with histological features.

  • Immunohistochemical analysis of Human malignant melanoma tissue, staining Pax2 with unpurified ab79389.
    Antigen retrieval was performed via heat mediation in a citrate buffer (pH 6). Sections were blocked with 0.1% Triton X-100/PBS containing 1% BSA and 10% horse serum for 1 hour. Samples were incubated with primary antibody overnight at 4°C. A Cy3®-conjugated goat anti-rabbit IgG was used as the secondary antibody.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of (A) human fetal kidney, (B) human normal kidney and (C) human renal cell carcinoma tissues labelled Pax2 with unpurified ab79389 at a dilution of 1/1000.

  • Immunocytochemistry/Immunofluorescence analysis of HEK293 cells labelling Pax2 with purified ab79389 at a dilution of 1/50. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

    Control 1: primary antibody (1/50) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

  • Anti-Pax2 antibody [EP3251] (ab79389) at 1/1000 dilution (purified) + Human fetal kidney tissue lysate at 20 µg

    Secondary
    HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/1000 dilution

    Predicted band size: 45 kDa
    Observed band size: 45 kDa



    Blocking and dilution buffer: 5% NFDM/TBST
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling Pax2 with purified ab79389 at a dilution of 1/500. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling Pax2 with purified ab79389 at a dilution of 1/500. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebral cortex tissue labelling Pax2 with purified ab79389 at a dilution of 1/500. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Flow Cytometry analysis of K562 cells labelling Pax2 with purified ab79389 at a dilution of 1/50 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

  • Anti-Pax2 antibody [EP3251] (ab79389) at 1/10000 dilution (unpurified) + Human fetal kidney tissue lysate at 10 µg

    Secondary
    HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

    Predicted band size: 45 kDa
    Observed band size: 45 kDa

  • Overlay histogram showing HEK293 cells stained with unpurified ab79389 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab79389, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a significantly decreased signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD

References

This product has been referenced in:
  • Jahangiri R  et al. PAX2 expression is correlated with better survival in tamoxifen-treated breast carcinoma patients. Tissue Cell 52:135-142 (2018). IHC-P ; Human . Read more (PubMed: 29857823) »
  • Wu B  et al. Reconstructing Lineage Hierarchies of Mouse Uterus Epithelial Development Using Single-Cell Analysis. Stem Cell Reports 9:381-396 (2017). IHC-P ; Mouse . Read more (PubMed: 28625536) »
See all 20 Publications for this product

Customer reviews and Q&As

1-8 of 8 Q&A

Answer



According to our records we have not received a customer report with similar results.
You have tried quite a few optimizations, but the result is still not satisfying.

Could you please let me know the species of your samples?
The antibody has been tested with human and mouse samples and we would guarantee it for these species.

If you have used any of the tested species, I would suggest either trying another lot or a different Pax2 antibody. A credit or refund can also be issued.

Please let me know your order or PO number in this regard as well as how you would like to proceed.

Read More

Question

WB Questionnaire
1) Abcam product code ab
EP3251, Ab79389
2) Abcam order reference number or product batch number
Lot:

3) Description of the problem
I just used Pax 2 primary antibodies in 1:5000 in western blot and
did not detect any band of 45kDa,
instead , I did detect a band of about 15kDa.
is there any isoform of Pax2 in this molecular weight as you may be
aware of (truncated, phosphorylated, ...?
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…):
isolated proteins from mice kidney tissues
Lysis buffer : RIPA
Protease inhibitors: 1x complete mini tablet (Roche)
Phosphatase inhibitors
Reducing agent
Boiling for ≥5 min? yes
Protein loaded ug/lane or cells/lane: 40ug
Positive control: a-tubulin
Negative control
5) Percentage of gel: 12%
Type of membrane : nitrocellulose membrane
Protein transfer verified: membrane staining
Blocking agent and concentration: 5% skim milk in 1xT-TBS
Blocking time : 2 hrs
Blocking temperature : Room tem.
6) Primary antibody (If more than one was used, describe in
“additional notes”) : Pax2
Concentration or dilution 1:5000
Diluent buffer : 5% skim milk in 1xT-TBS
Incubation time: overnight
Incubation temperature: 4C
7) Secondary antibody: anti-rabbit
Species: rabit
Reacts against: mouse
Concentration or dilution : 1:5000
Diluent buffer : 5% skim milk in 1xT-TBS
Incubation time: 2 hrs
Incubation temperature: room temp
Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies:
Buffer: 1xT-TBS
Number of washes: 3
9)Detection method: ECL
10) How many times have you run this staining? 1
Do you obtain the same results every time?
What steps have you altered to try and optimize the use of this antibody?

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab79389 is tested and covered by our 6 month guarantee for use in WB and mouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab79389:

Pax2 is a nuclear protein and therefore I suggest to check if the RIPA buffer has enough SDS to lyse the nucleus. The SDS percentage might need to be optimised.

To my knowledge the best expression of Pax2 is in fetal kidney and the expression level in adult kidneymight not be high enough to see the protein on a WB. I therefore recommend to use fetal kidney extract or to prepare a nuclear fraction in order to enrich nuclear protein when running a WB.

When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : https://www.abcam.com/ab9385 .

I could not find any shorter isoforms than 42kDa, but according to genecards (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PAX2&search=Pax2+) there are alternatively spliced variants which could be short enough to show up as ca 15kDa.

I think this this results indicates no specific band is found and that when using fetal kidney samples this will be remedied.

Please confirm that the correct secondary antibody was used. It should be an anti rabbit antibody from a species other than rabbit.


I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again.

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Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.

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Answer

Thank you again for your email.
The concentration of your lot is 0.2290 mg/ml.

Please let me know if you have anymore questions.

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Answer

In order to help you, I would need to know which lot./ batch number you received from us?

I am looking forward to your reply.

Read More

Answer

According to our records, ab79839 was proving difficult to use inWB and we were in contact in order to help resolve the issue.

Looking at our correspondence, it appears that we are awaiting more details in order to help us better understand the difficulties experienced. If the requested information has already been sent, it appears that it did not reach our Scientific Support team and we apologize for this inconvenience. In this case we would like to ask for the information again so that we can reach a resolution.

If the issue has already been settled, please let us know so that we can be assured that the problem has been solved to your satisfaction and update our records.

We wish you the best of luck with your research and look forward to a reply.

Read More

Answer

Thank you for your email. I am sorry to hear that you are having trouble using this antibody. We have sold many units of this antibody without any complaint so I am wondering if you could provide more details about the protocol used. This will help us to check if the protocol used was fine. I have attached a questionnaire. Please fill in all the information. Incase the protocol usedis fineand no further optimization could be done; I will then provide the replacement antibody. We will look forward receiving your reply soon.

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Answer

Thank you for your enquiry. I am sorry to confirm we are not able to provide this antibody BSA free. This particular antibody is sourced externally. With externally sourced products, they are often available for sale by several companies. I am sorry that for commercial reasons we are not able to provide the name of our supplier. I can suggest you may like to try one of the purification kits from the catalog.  You could consider tying this to remove the BSA from the antibody solution. The following kits are suitable for tissue culture supernatant. I can recommend to review the online datasheets for further information: ab109206 Antibody TCS Purification Kit (1 purification) Click here (or use the following: https://www.abcam.com/index.html?datasheet=109206). ab109207 Antibody TCS Purification kit (3 purifications) Click here (or use the following: https://www.abcam.com/index.html?datasheet=109207). I am sorry we are not able to provide exactly what you require on this occasion, but hope this wil be helpful. If you have any further questions, please do not hesitate to contact us.  

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